CN116529599A - In vitro method for detecting SARS-CoV-2in oral cavity sample using colorimetric immunosensor and related colorimetric immunosensor - Google Patents

In vitro method for detecting SARS-CoV-2in oral cavity sample using colorimetric immunosensor and related colorimetric immunosensor Download PDF

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CN116529599A
CN116529599A CN202180078534.4A CN202180078534A CN116529599A CN 116529599 A CN116529599 A CN 116529599A CN 202180078534 A CN202180078534 A CN 202180078534A CN 116529599 A CN116529599 A CN 116529599A
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斯特凡尼娅·阿莫罗西
安东尼奥·切尔沃
巴尔托洛梅奥·德拉·文图拉
埃琳娜·索切利
萨尔瓦多·塞索
拉斐尔·韦洛塔
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Abstract

An in vitro assay method for detecting SARS-CoV-2 virions in oral biological samples selected from saliva and sputum using a colorimetric immunosensor, and related kits, are described herein. The methods and kits of the present invention are based on the use of Jin Buhuo nanoparticles that carry on their surface at least one antibody capable of binding to the SARS-CoV-2 surface antigen.

Description

In vitro method for detecting SARS-CoV-2in oral cavity sample using colorimetric immunosensor and related colorimetric immunosensor
Technical Field
The present invention relates to an in vitro method for detecting SARS-CoV-2in oral biological samples by using a colorimetric immunosensor and a related colorimetric immunosensor.
Statement of technology
In the research and diagnostic field, colorimetry-type optical biosensors are becoming increasingly important due to their versatility, ease of use and ability to reach very low detection limits (limit of detection, LOD) of analytes.
Gold nanoparticle-based colorimetric biosensors are known in optical biosensors, which utilize the physical properties of gold nanoparticles, called localized surface plasmon resonance (Localized Surface Plasmon Resonance, LSPR) to monitor color changes when different-sized clusters are formed after interactions between analytes to be detected and gold nanoparticles.
Optical transducers (transducers) are of particular interest for direct detection of microorganisms (no label). These sensors are designed to detect the minimum shift in refractive index or thickness that occurs when a cell attaches to a receptor immobilized on the surface of a transducer, correlating changes in concentration, mass or number of molecules with direct changes in light characteristics. The sensing mechanism is based on the conversion of a signal generated by binding to the target to be detected into a physical signal that can be amplified and detected. Nanomaterials are characterized by extremely small dimensions and can be suitably surface modified, which allow highly specific interactions with biomolecular targets, thus showing great potential in the field of biological detection.
More generally, gold nanoparticles find application in a wide range of disciplines including magnetic fluids, catalysis, biotechnology/biomedical, magnetic resonance imaging and environmental remediation. In most of these applications, better functionality of these nanoparticles is observed when their size is below a critical value, which depends on the material, but is typically about 10-20nm.
Biosensors for determining an analyte of interest based on the specificity of the interaction between an antigen and a corresponding antibody are commonly referred to as immunosensors. Antibody immobilization procedures are a critical step in the construction of these devices because the orientation of the antibody molecules on the electrode surface significantly affects the performance of the biosensor. In fact, the formation of an antibody layer with its binding sites well oriented and facing the antigen improves the efficiency of the biosensor, making the choice of immobilization method one of the most important aspects to be considered in the construction of immunosensors. Generally, antibody immobilization methods involve physical or chemical adsorption of these molecules.
In the simplest adsorption procedure, mention is made of methods which use ionic or electrostatic bonds between the antibody and the surface, hydrophobic interactions and van der Waals bonds, and do not require chemical modification of the protein (Shalma, byrne and Kennedy,2016; um et al 2011). The main disadvantage of the above method is that the antibodies are randomly oriented and thus may not be able to properly expose the antigen binding site.
A more efficient method of immobilizing antibodies is based on the formation of covalent bonds between the antibodies and the gold surface (Alves, kiziltepe and Bilgicer,2012; ho et al, 2010; vashist et al, 2011; rahman et al, 2007). For example, biotinylated antibodies may be immobilized on surfaces modified with streptavidin or avidin (Barton et al, 2009; ouerghi et al, 2002) or antibodies may be immobilized on surfaces modified with proteins (such as protein A or protein G) (J.E. Lee et al, 2013; inkpen et al, 2019; sharafeldin and rushing, 2019; fowler, stuart and Wong, 2007). Finally, immobilization methods involving antibodies captured in a polymer matrix have been developed in the last decade (Sun et al, 2011; bereli et al, 2013; moschallski et al, 2013; yamazole, 2019).
Among the possible immobilization strategies, the formation of self-assembled monolayers (self-assembled monolayer, SAM) is one of the most widely used methods for constructing immunosensors. For example, the immobilization of the orientation of the antibody on the gold surface of the electrode can be achieved by the formation of SAMs using thiolcarboxylic acids (Barreiros dos Santos et al, 2013; malvano, piclloton and Albane, 2018; wan et al, 2016) or by immobilization of the antibody on an electrochemically deposited cysteamine layer (Malvano, pilloton and Albane, 2018). Furthermore, the use of cross-linking agents such as glutaraldehyde has recently been reported, in particular for immobilization of anti-E.coli antibodies on polyaniline substrates, with interesting results in the detection of such bacteria (Chodwhury et al 2012). SAM is therefore widely used as a linker for immobilizing antibodies on gold surfaces in an aligned manner, and although they show many advantages in various applications, there are still several aspects to consider in order to understand and control their physical and chemical properties (Verica et al, 2010; mandler and Kraus-Ophir,2011; chaki and Vijayamohanan, 2002). The self-assembled monolayer on the gold surface is typically represented as a perfect monolayer in which the molecules are in a perfect packaging configuration (packed configuration). In fact, this idea is far from realistic and quality control of SAM is a key point in many applications. The construction of well assembled monolayers is strongly dependent on the purity of the chemicals and solutions used and even the presence of minimal amounts of contaminants, such as thiolated molecules, which are typical impurities in thiol compounds, may lead to non-uniform and thus non-ideal layers (c.y.lee et al 2005).
In recent years, different types of immunosensors have been described in studies published in the literature.
Iarossi, M et al (2018) ("Colorimetric Immunosensor by Aggregation of Photochemically Functionalized Gold Nanoparticles" ACS Omega 3,4,3805-3812) describe a colorimetric immunosensor that utilizes the phenomenon of surface plasmon resonance of gold nanoparticles, and the use of such a system for detecting human IgG immunoglobulins.
Liu Y et al (2015) ("Colorimetric detection of influenza A virus using antibody-functionalized gold nanoparticles" analysis 140 (12) 3989-3995) studied the use of colorimetric immunosensors based on gold nanoparticles modified with anti-hemagglutinin monoclonal antibodies to determine influenza A virus. However, no evidence of the clinical efficacy of immunosensors is provided in this document.
In Della Ventura B et al (2020) ("Colorimetric Test for Fast Detection of SARS-CoV-2in Nasal and Throat Swabs"MedRixv doi:https://doi.org/10.1101/ 2020.08.15.20175489and ACS Sensors 5,3043-3048) relates to the use of colorimetric immunosensors for detecting SARS-CoV-2 coronavirus in nasopharyngeal swabs.
The ongoing severe epidemic caused by the new severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has created major public health challenges in many countries. Because of the lack of specificity of the symptoms of severe disease caused by the new coronavirus known as covd-19, validation of diagnosis requires laboratory tests to be performed on respiratory and/or serum samples from patients. Large scale diagnostic tests also play a key role in isolating asymptomatic covd-19 patients in an attempt to prevent the spread of infection.
In the current procedure for diagnosing SARS-CoV-2 coronavirus infection, a reverse transcription polymerase chain reaction (reverse transcription polymerase chain reaction, RT-PCR) based method plays a major role. This method allows the identification of viral genomes in samples from the upper respiratory tract, in particular samples obtained using nasopharyngeal swabs. However, diagnostic applications of PCR have major limitations due to the complexity of execution and timing, the need for specialized instrumentation and trained personnel.
Immunological methods for diagnosing covd-19 have also been developed to present significant problems. For example, lateral flow immunochromatographic assays are characterized by low sensitivity in cases that allow for rapid analysis of many samples.
Other problems associated with methodologies aimed at diagnosing SARS-CoV-2 infection involve selecting the most appropriate sample to find the viral particle, more specifically the intact particle. Among them, the currently selected samples are represented by respiratory tract samples, in particular nasopharyngeal swabs. However, such sampling requires a precise procedure, that is, to be effective, sampling from the nose must be performed by pushing the swab down to the pharynx rather than toward the nasal cavity.
Thus, there is a need to provide diagnostic tests that allow for rapid identification of SARS-CoV-2 virus by simple procedures while maintaining high specificity and sensitivity parameters.
These and other objects are achieved by an in vitro method and related kit as defined in the appended independent claims, which are suitable for detecting SARS-CoV-2 virions (virions) in an oral biological sample of a subject.
Additional features of the invention are defined in the dependent claims, which form an integral part of the description.
As will be clear from the detailed description below, the in vitro method according to the invention allows to obtain diagnostic results in a very short time, in a very small range, and requires a minimum amount of sample to be analyzed in the range of about one milliliter of volume, which is very easy to collect. This procedure is particularly simple and does not require any complex instrumentation, which also allows a significant cost reduction.
Accordingly, a first object of the present invention is an in vitro method for detecting SARS-CoV-2 virions in an oral biological sample of a subject, the oral biological sample being selected from saliva and sputum, the method comprising the steps of:
a) Contacting the biological sample with a colloidal suspension of gold-capturing nanoparticles carrying on their surface at least one antibody capable of binding to a SARS-CoV-2 surface antigen selected from the group consisting of: membrane protein (M), envelope protein (E), spike protein (S), and any combination thereof;
(b) Determining the formation of gold nanoparticle clusters on the surface of the SARS-CoV-2 virion in the reaction mixture, said clusters resulting from the interaction between the antibody and the antigen, the determining being performed by detecting a change in an optical parameter of the reaction mixture, said change in the optical parameter of the reaction mixture being indicative of the presence of the SARS-CoV-2 virion in the oral sample.
Within the scope of the present specification, the term "virosome" refers to a mature viral particle, including its genome, nucleocapsid (nucleocapsid) and envelope.
The method according to the invention is based on the physical principle of Localized Surface Plasmon Resonance (LSPR), which consists in the occurrence of coherent and non-propagating oscillations of free electrons in metal particles after irradiation with electromagnetic waves whose frequency resonates with the surface plasmon. The resonance of the surface plasmons (which imparts color to the colloidal solution) depends on various factors, such as the size of the nanoparticles, and can vary significantly when the nanoparticles come into contact with each other or in any case at distances much smaller than their own diameter. In general, the coupling of metal nanoparticles (such as gold nanoparticles) to each other in colloidal suspensions occurs by forming dimers, trimers or larger chains until clusters are formed, and involves a change in plasmon resonance, and thus a change in solution color, which can even be detected with the naked eye.
According to the invention, the clustering (aggregation) of gold nanoparticles in the reaction mixture occurs mediated by a biological mechanism consisting in a specific interaction between the antibodies immobilized on the surface of said capture nanoparticles and the corresponding antigens present on the surface of the SARS-CoV-2 virus. Thus, clustering only occurs when SARS-CoV-2 virus particles are present in the test sample, thus providing great specificity to the methods of the invention.
Oral samples suitable for use in the methods of the invention are selected from saliva and sputum.
As described in more detail in the experimental section below, the inventors surprisingly found that the method of the invention allows for the detection of SARS-CoV-2 virus in saliva or sputum of a subject. Although this type of ease of sampling has undoubted advantages, their use in the method as defined previously has been particularly difficult to date, if not impossible, not only because of the high salt concentration in these samples that would cause non-specific clustering of gold nanoparticles, making the biosensor unusable, but also because of the presence of mucus (mucus). The latter, although not affecting the kinetics of clustering, contains opaque species that alter the absorption spectrum. Unfortunately, the variability of mucosal concentration (not only in humans, but also for the same person over time) makes it impossible to normalize the contribution of mucus to optical readings. In addition, saliva contains high concentrations of proteins that may interfere with antigen-antibody interactions. All of these considerations highlight the properties of the saliva matrix compared to other virosome-containing solutions, e.g. solutions composed of viral transport vehicles (Virus Transport Medium, VTM). To demonstrate the importance of the properties of each matrix, this makes it almost impossible to immediately extend the analytical technique from one matrix to another, enough to consider the "gold standard" technique given by RT-PCR ineffective on saliva matrices, but rather the need to immerse the nasopharyngeal swab in the VTM.
As described above, the metal nanoparticles used in the method according to the present invention are gold nanoparticles.
Preferably, the gold nanoparticles have a diameter in the range of 1nm to 100nm, more preferably 2nm to 40 nm. Most preferred are gold nanoparticles having a diameter of 20nm.
In the method according to the invention, the at least one antibody on the surface of the capture gold nanoparticle is capable of binding to a SARS-CoV-2 surface antigen selected from the group consisting of: membrane protein (M), envelope protein (E), spike protein (S), and any combination thereof.
Together, the above viral proteins contribute to the formation of the outer viral envelope, as is known in the art. Wherein the spike protein binds SARS-CoV-2 to the host cell by promoting fusion of the viral envelope with the cell membrane.
Among the antibody molecules suitable for use in the method according to the invention, mention is made, as non-limiting examples, of monoclonal or polyclonal antibodies, monomeric (Fab) or dimeric (F (ab') 2) antibody fragments, single chain antibody fragments (single chain antibody fragment, scFv) or any binding protein derived from an antibody scaffold (antibody scaffold).
According to the present invention, it is contemplated that the anti-SARS-CoV-2 antibody as defined above can be present on the capture gold nanoparticle in any possible combination. The proximity of the antibody to the gold nanoparticle surface allows the distance between the nanoparticles of the clusters to be minimized, allowing LSPR phenomena to occur.
Methods suitable for achieving immobilization of one or more antibody molecules in close proximity to gold nanoparticle surfaces are known in the art, even though they are essentially limited to physical adsorption or photochemical techniques (photo chemical immobilization techniques, PIT) that achieve immobilization of the antibody on gold surfaces in the correct orientation by irradiation of these molecules with ultraviolet light [ Della venturi b.et al (2019) "Biosensor surface functionalization by a simple photochemical immobilization of antibodies: experimental characterization by mass spectrometry and surface enhanced Raman spectroscopy". Analyst 144,6871-6880].
Unlike physical adsorption, the PIT process allows robust and durable functionalization (functionalization), allowing industrial application of such a process.
The selection of the most suitable antibody immobilization method within the scope of the present invention is well within the skill of one of ordinary skill in the art.
Preferably, the amount of Jin Buhuo nanoparticles as defined above in the colloidal suspension is between 1 and 10 based on the total volume of the suspension 20 Nanoparticles (np)/ml, more preferably 10 to 10 15 np/ml. In an even more preferred embodiment, the amount of Jin Buhuo nanoparticles is 10 based on the total volume of the suspension 10 np/ml。
Advantageously, the method according to the invention allows for the complete detection of SARS-CoV-2 virus particles in a test sample due to the ability of gold nanoparticles carrying antibodies specific for viral surface proteins to cluster on the surface of the virion by being held very close to each other and forming a layer around the particles. Thus, due to the specific assay of active viral particles, the method according to the invention is particularly suitable for identifying cases in which SARS-CoV-2 infection is still ongoing.
According to an embodiment, the method of the invention further comprises the step of filtering the reaction mixture obtained in step a) by using a filter element having pores with a diameter of 35 to 65 micrometers (μm), preferably 40 to 60 μm, more preferably 45 to 55 μm, for example having pores with a diameter of 45, 46, 47, 48, 49, 50, 51, 52, 53, 54 or 55 μm.
Within the scope of the present invention, the above-described filtration step removes or significantly reduces the mucosal components of saliva or sputum samples present in the reaction mixture, thereby increasing the sensitivity and specificity of the method of the present invention.
As described above, in the method according to the present invention, the clustering of gold nanoparticles on the surface of SARS-CoV-2 virion is determined by detecting the change in the optical parameter of the reaction mixture.
In one embodiment of the method of the invention, the detected change in the optical parameter is a visually detectable change in the color of the reaction mixture.
In this embodiment, a further optional step consists in comparing the detected color of the reaction mixture with a colorimetric scale (colorimetric scale). This step improves the interpretation quality of the results (interpretative quality).
It is also important and advantageous that the above embodiments do not require the use of instrumentation.
In another embodiment of the invention, the detected change in the optical parameter is a decrease in the transmittance value (transmittance value) of the reaction mixture measured at a predetermined wavelength (preferably 560 nm) in the visible range.
Within the scope of the present description, the expression "wavelength in the visible range" refers to a wavelength between about 390nm and about 760 nm.
According to the above embodiment, the measurement of the transmittance value of the reaction mixture may be performed by using a photometer or a colorimeter instrument, which is preferably calibrated with a standard solution having a transmittance value of 100%.
In a further embodiment of the invention, the detected change in the optical parameter is an increase in the absorbance value of the reaction mixture measured at a wavelength in the predetermined visible range, preferably at 560 nm. A suitable instrument for performing the absorbance measurement described above is a spectrophotometer, which may be, for example, a portable spectrophotometer or a bench-top spectrophotometer.
According to another embodiment, the detected change in the optical parameter is an increase in area under the absorption spectrum of the reaction mixture in the wavelength range between 200nm and 700 nm.
In this embodiment, the method according to the invention allows for a quantitative determination, which is indicative of the SARS-CoV-2 viral load in the test sample. According to this embodiment, an "absolute" measure of viral load can also be obtained using the standard curve in addition.
In yet another embodiment, the method of the invention is in a competitive format wherein the change in the optical parameter is an increase in absorbance value of the reaction mixture measured at a wavelength in the range of 600nm to 700 nm.
This competitive embodiment involves the use of high salts and is therefore a particularly unstable colloidal suspension of gold-capturing nanoparticles. More specifically, according to such an embodiment, the colloidal suspension comprises a salt selected from the group consisting of: sodium citrate, sodium chloride, potassium phosphate, sodium phosphate, calcium chloride, potassium chloride, and any combination thereof, and the above salts are present in the colloidal suspension at a concentration in the range of 150 millimoles (mM) to 250 mM.
Within the scope of the present invention, the salt is preferably sodium citrate, more preferably sodium citrate present in the colloidal suspension at a concentration of 160 mM.
The presence of the salt results in the addition of saliva that is free of and therefore not infected by the SARS-CoV-2 virus to induce the gold-capturing nanoparticles to cluster with each other in clusters, resulting in a macroscopic color change in the reaction mixture. On the other hand, if saliva contains SARS-CoV-2 virus particles, the formation of clusters of functionalized gold nanoparticles on the virosome surface prevents the nanoparticles from clustering with each other. Since the clustering of gold nanoparticles on the virosome surface results in a shift of the formants in the absorbance spectrum of the reaction mixture that is different from the shift caused by salt-induced clustering in the absence of virosomes, more precisely from a wavelength of about 560nm to a wavelength in the range of 600 to 700nm, the presence of virosomes in the oral sample comprises a slight change in color compared to the very bright color change observed in the absence of virosomes.
As mentioned before, kits comprising means suitable for performing the method according to the invention are also included within the scope of the invention.
Accordingly, a second aspect of the present invention is a diagnostic kit for detecting SARS-CoV-2 virions in an oral biological sample of a subject, the oral biological sample selected from saliva and sputum, the diagnostic kit comprising a colloidal suspension of Jin Buhuo nanoparticles carrying on their surface at least one antibody capable of binding to a SARS-CoV-2 surface antigen selected from the group consisting of: membrane protein (M), envelope protein (E), spike protein (S), and any combination thereof.
According to an embodiment, the colloidal mixture of Jin Buhuo nanoparticles comprises a salt selected from the group consisting of: sodium citrate, sodium chloride, potassium phosphate, sodium phosphate, calcium chloride, potassium chloride, and any combination thereof, the salt being present in the colloidal suspension at a concentration in the range of 150 millimoles (mM) to 250 mM.
Within the scope of the present invention, preferably the salt is sodium citrate, more preferably sodium citrate present in the colloidal suspension at a concentration of 160 mM.
In one embodiment, the diagnostic kit of the invention further comprises a support comprising a colorimetric scale, such as a colorimetric strip.
In another embodiment, the diagnostic kit of the present invention further comprises a portable colorimeter or photometer.
Preferably, the portable photometer is equipped with a tungsten filament lamp and a monochromator (monochromator) capable of isolating the wavelength at 560 nm.
Preferably, the portable colorimeter is equipped with a diode capable of emitting light at 560 nm.
Among the portable instruments suitable for use in the kit of the invention, mention is made, as examples, of the portable type HI96759 photometer and the portable type HI759 colorimeter, both of the Karana instruments company (Hanna Instruments).
In the diagnostic kit of the present invention, a colloidal suspension comprising Jin Buhuo nanoparticles can be dispensed into a plurality of individual disposable tubes (single disposable test tubes).
Alternatively, the colloidal suspension may be provided in a single package, for example in a dedicated dropper apparatus.
In another embodiment, the diagnostic kit of the invention further comprises a filter element as defined hereinbefore with respect to the method of the invention.
According to this embodiment, the filter element may be housed inside the disposable test tube, preferably near one end of the body of the test tube opposite to the end where the colloidal suspension is introduced.
Within the scope of the present invention, the filter element in the kit is preferably a hydrophilic polyethylene filter.
The following experimental examples are provided for illustrative purposes only. Reference is made herein to the accompanying drawings, in which:
FIG. 1 is a schematic representation of the method of functionalizing gold nanoparticle surfaces with G-type immunoglobulins (photochemical immobilization technique, PIT). The IgG antibody was irradiated with ultraviolet light using a lamp of appropriate power to reduce disulfide bridge (disulfide bridge) at a specific position of the light chain constant portion of the antibody. The generation of thiols allows the formation of covalent bonds between the antibody and the gold nanoparticle surface, freeing one of the two antigen recognition portions of the antibody.
Figure 2 is a schematic representation of the process of the invention. The colloidal suspension of gold nanoparticles functionalized with anti-SARS-CoV-2 antibody is contacted with a sample containing virus to form a reaction mixture. After the functionalized gold nanoparticles cluster around the viral particles, the reaction mixture changes color. As the concentration of virus particles increases, the shift toward blue increases.
Experimental part
Example 1: preparation of colloidal gold solution (Synthesis of nanoparticles)
For their experiments, the inventors used a protocol known in the art (turkivich's prescriptionMethod) yields a synthesis of gold nanoparticles of about 20nm diameter. According to this protocol, gold is first dissolved in water and sodium citrate is added to cause the reduction of gold such that gold seeds are produced and then gold grows around the gold seeds. The synthesis reaction involved mixing 1mL of HAuCl in 100mL of MilliQ (ultrapure) water 4 (10 mg/mL) and 2mL sodium citrate dihydrate (25 mg/mL). The operating temperature was maintained at 90 ℃ with gentle stirring. The formation of gold nanoparticles was identified by a dramatic change in solution color from yellow to orange.
At the end of the synthesis, the solution was centrifuged at 6G for 30 minutes, thereby obtaining gold nanoparticles to be functionalized.
Example 2: functionalization
The surface of the gold nanoparticles is functionalized by using a mechanism called Photochemical Immobilization Technology (PIT) as described in fig. 1.
Briefly, igG antibodies (0.1 mg/mL) directed against the membrane protein (membrane, M), envelope protein (envelope, E) and spike protein (S) of the SARS-CoV-2 virus were used.
A quartz cuvette containing an antibody solution at a concentration of 1. Mu.g/mL was inserted into a specially designed UV lamp and irradiated with UV radiation for 30 seconds in order to reduce some disulfide bonds at specific positions of the antibody. Subsequently, gold nanoparticles having a diameter of 20nm were functionalized to obtain anti-envelope antibody having a nanoparticle concentration of 10 10 Nanoparticle (np)/mL, nanoparticle concentration of anti-spike antibody of 10 10 nanoparticle concentration of np/mL and anti-membrane antibody 10 10 np/mL. Any empty space left on the gold nanoparticles was then blocked by using a solution containing BSA (50. Mu.g/mL).
Finally, colloidal suspensions containing three different antibodies were mixed together in a 1:1 ratio to obtain a single suspension of gold nanoparticles carrying three anti-SARS-CoV-2 antibodies, thereby significantly increasing the specificity of the system.
Purification of the resulting samples was performed by centrifugation at 6G for 10 minutes.
Example 3: sample preparation
Saliva samples were collected using a swab and immediately transferred to a colloidal suspension of functionalized gold nanoparticles having a volume of 0.5 ml. Any virions on the swab were released by vigorously spinning the swab in suspension about its own axis for about 10 seconds. The inventors found that, unlike prior art assays, the method according to the invention surprisingly does not require re-suspending the saliva sample in a buffer solution after sampling. The inventors have also found that other stirring methods (e.g. stirring the test tube up and down in an uncoordinated manner) do not achieve the same results in the same time, and also show a lack of reproducibility and reproducibility.
Optionally, to remove mucosal components present in the sample, the reaction mixture obtained by mixing saliva or sputum samples with a colloidal suspension of gold capture nanoparticles is dropped through a hydrophilic polyethylene filter with 50 micron diameter pores and collected in a cuvette below. The time taken for this process varies over an interval of 3-10 minutes, depending on the viscosity of the saliva. After filtration, the reaction mixture was read.
Example 4: detection of
Gold capture nanoparticles prepared and functionalized as described above were used in SARS-CoV-2 virus detection experiments.
Briefly, after mixing saliva or sputum samples with colloidal suspensions of gold-capturing nanoparticles, a color change occurs in the reaction mixture, from orange to blue to purple. Specifically, as the virus concentration increases, the shift toward blue increases. A simple buffer solution in which no color change was seen was used as a negative control.
For transmittance or absorbance analysis, after resuspension, a defined volume of saliva or sputum sample was placed into a test tube (wheatstone type, wheaton type) already containing a gold capturing nanoparticle suspension by means of a sterile disposable Pasteur pipette.
Alternatively, a defined volume of test sample is dispensed into a supplied cuvette together with a gold capturing nanoparticle suspension.
The reaction mixture obtained by the above preparation method is mixed by stirring a test tube/cuvette, or by pipetting, thereby allowing clusters of functionalized gold nanoparticles to be formed on the surface of SARS-CoV-2 virions.
Subsequently, optionally after filtration, the test tube or cuvette is placed in a portable reader provided that provides an absorbance or transmittance value indicative of sample positivity/negativity, i.e., the presence or absence of SARS-CoV-2 virus particles.
In experiments conducted by the present inventors, a portable photometer equipped with a tungsten lamp and a monochromator capable of isolating the wavelength at 560nm after proper calibration with a standard was used. A simple "blank" (i.e., a sample that specifies 100% transmittance or 0% absorbance) was used as a standard. Disposable cuvettes containing the reaction mixtures were used for photometric analysis.
In their experiments, the inventors optionally used a colorimeter device equipped with a diode capable of emitting light at 560nm and returning transmittance values similar to the devices described above. In this program mode, after resuspension, saliva or sputum samples are dispensed and assayed directly in disposable reaction tubes pre-packaged with colloidal suspensions of gold capture nanoparticles.
The transmittance values measured in the above experiments indicate the amount of SARS-CoV-2 virus particles present in the test sample.
To measure the absorbance of the reaction mixture, the inventors have also used a bench-top spectrophotometer instrument capable of emitting in a broad spectrum of wavelengths, for example in the range of 200nm to 700 nm. Absorbance measurements at different wavelengths allow the area under the absorbance spectrum to be calculated, providing a "relative" quantitative determination of viral load. By calibration of the described technique, the viral load measurement can become "absolute".
Table 1 below shows the results of 6 patients (3 positive and 3 negative) obtained using the PCR method and the method of the present invention in parallel. For PCR analysis, the data is expressed as cycle threshold (Ct) corresponding to the cycle of the PCR reaction in which the emitted fluorescence exceeds the threshold. For analysis according to the method of the invention, the data are expressed as absorbance values (Abs). The term "X" denotes a negative sample.
TABLE 1
PCR threshold (Ct) Abs at 560nm
18 0.325
22 0.294
35 0.224
x 0.171
x 0.195
x 0.176
Table 1 shows that the negative samples gave absorbance values of 0.18.+ -. 0.01. Even considering the 3SD standard, the sample with the lowest viral load (ct=35) is clearly distinguished from the negative sample. In fact, PCR thresholds above 35 are considered negative. These results demonstrate that the method according to the invention has detection limits comparable to PCR.
Example 5: method of the invention with competitive configuration
Experiments conducted with a competitive configuration using the method of the present invention resulted in particularly unstable colloidal suspensions of captured nanoparticles. For this purpose, the following two steps are carried out: 1) Sodium citrate at a concentration of 160mM is used during the step of production in water; 2) Excess reagent was removed by centrifugation at 4.5G for 30 minutes and the pellet was resuspended in an aqueous solution containing 160mM sodium citrate. Subsequently, saliva containing SARS-CoV-2 virus particles is added to the colloidal suspension of captured nanoparticles obtained as described above, such that the shift of the formants in the absorption spectrum of the reaction mixture is different from the shift caused by the addition of the uninfected saliva reaction mixture, thus, wherein the clustering of the nanoparticles is induced only by the salt. In fact, the inventors observed very bright color changes in the reaction mixture in the absence of virions, which demonstrated a slight color change in the presence of virus.
In the directly configured process of the invention as described in example 4, an absorption peak of the reaction mixture was observed at a wavelength of about 560nm, whereas in the competitively configured process of the invention, a much larger peak of the reaction mixture was observed at a wavelength in the visible range of 600-700 nm.
Table 2 below shows the results of 6 patients (3 positive and 3 negative) obtained using the PCR method and the method of the present invention in parallel. The data are presented above with reference to table 1. The term "x" denotes a negative sample.
TABLE 2
PCR threshold (Ct) Abs at 560nm Abs at 600nm
15 0.274 0.157
19 0.252 0.133
22 0.214 0.122
x 0.199 0.046
x 0.187 0.052
x 0.192 0.064
The results shown in Table 2 demonstrate that there is a measurable difference between the samples containing SARS-CoV-2 (positive cases) and the samples without virus (negative cases).

Claims (16)

1. An in vitro method for detecting SARS-CoV-2 virions in an oral biological sample of a subject, the oral biological sample selected from saliva and sputum, the method comprising the steps of:
a) Contacting the biological sample with a colloidal suspension of gold capturing nanoparticles carrying on their surface at least one antibody capable of binding to a SARS-CoV-2 surface antigen selected from the group consisting of: membrane protein (M), envelope protein (E), spike protein (S), and any combination thereof; and
(b) Determining the formation of clusters of gold nanoparticles on the surface of the SARS-CoV-2 virion in the reaction mixture, the clusters resulting from the interaction between the antibody and the antigen, the determination being made by detecting a change in an optical parameter of the reaction mixture, the change in the optical parameter of the reaction mixture being indicative of the presence of the SARS-CoV-2 virion in the oral biological sample.
2. The method of claim 1, wherein the change in the optical parameter is a change in color of the reaction mixture detectable by the naked eye.
3. The method of claim 2, further comprising the step of comparing the detected color of the reaction mixture to a colorimetric scale.
4. A method according to claim 1, wherein the change in the optical parameter is a decrease in the transmittance value of the reaction mixture measured at a predetermined wavelength in the visible range, preferably at 560 nm.
5. A method according to claim 1, wherein the change in the optical parameter is an increase in absorbance value of the reaction mixture measured at a predetermined wavelength in the visible range, preferably at 560 nm.
6. The method of claim 1, wherein the change in the optical parameter is an increase in area under the absorption spectrum of the reaction mixture in a wavelength range between 200nm and 700 nm.
7. The method of claim 1, in a competitive form,
wherein the colloidal suspension of Jin Buhuo nanoparticles comprises a salt selected from the group consisting of: sodium citrate, sodium chloride, potassium phosphate, sodium phosphate, calcium chloride, potassium chloride and any combination thereof, the salt being present in the colloidal suspension at a concentration in the range of 150 millimoles (mM) to 250mM, and
wherein the change in the optical parameter is an increase in absorbance value of the reaction mixture measured at a wavelength in the range of 600nm to 700 nm.
8. The method according to any one of claims 1 to 7, further comprising the step of filtering the reaction mixture obtained in step a) by using a filter element having pores with a diameter in the range of 35 to 65 micrometers (μιη).
9. A method according to any one of claims 4 to 8, wherein the change in the optical parameter is detected by means of a colorimeter or photometer.
10. A diagnostic kit for detecting SARS-CoV-2 virions in an oral biological sample of a subject, the oral biological sample selected from saliva and sputum, the diagnostic kit comprising a colloidal suspension of gold capture nanoparticles, the Jin Buhuo nanoparticles carrying on their surface at least one antibody capable of binding to a SARS-CoV-2 surface antigen selected from the group consisting of: membrane protein (M), envelope protein (E), spike protein (S), and any combination thereof.
11. The diagnostic kit of claim 10, wherein the colloidal mixture comprises a salt selected from the group consisting of: sodium citrate, sodium chloride, potassium phosphate, sodium phosphate, calcium chloride, potassium chloride, and any combination thereof, the salt being present in the colloidal suspension at a concentration in the range of 150 millimoles (mM) to 250 mM.
12. The diagnostic kit of claim 10 or 11, wherein the colloidal suspension is dispensed into a plurality of individual disposable tubes.
13. The diagnostic kit of any one of claims 10 to 12, further comprising a support comprising a colorimetric scale.
14. The diagnostic kit of any one of claims 10 to 12, further comprising a portable colorimeter or portable photometer.
15. The diagnostic kit of any one of claims 10 to 14, further comprising a filter element having pores with a diameter in the range of 35 to 65 micrometers (μιη).
16. The diagnostic kit of claim 15, wherein the filter element is a hydrophilic polyethylene filter.
CN202180078534.4A 2020-09-22 2021-09-21 In vitro method for detecting SARS-CoV-2in oral cavity sample using colorimetric immunosensor and related colorimetric immunosensor Pending CN116529599A (en)

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IT102020000022351 2020-09-22
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